Fitzgerald M M, Churchill M J, McRee D E, Goodin D B
Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Biochemistry. 1994 Apr 5;33(13):3807-18.
In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.
在细胞色素c过氧化物酶的氧化“ES”状态下，色氨酸-191被高价血红素可逆地氧化为稳定的阳离子自由基。为了探索在此位点构建杂环化合物结合位点的可能性，构建了突变体W191G。W191G的两个分辨率为2.1埃和2.3埃的独立晶体结构表明，W191G在色氨酸-191位点含有一个明确的、约180埃³的空腔。该空腔被五个有序水分子占据，这些水分子彼此之间、与极性主链原子以及与天冬氨酸-235的羧酸盐形成广泛的氢键网络。在筛选了多种杂环化合物后，有证据表明取代咪唑与W191G的空腔结合。用咪唑滴定W191G导致Soret吸收带发生扰动，而W191H、W191F或天然酶未观察到这种情况。在pH 6.0时，苯并咪唑、咪唑、2-乙基咪唑、1-甲基咪唑、2-甲基咪唑和1,2-二甲基咪唑与W191G结合的解离常数分别为2.58、0.70、0.36、0.057、0.047和0.027 mM。1,2-二甲基咪唑表现出最高的结合亲和力，表明空间相互作用和填充空腔的效率是特异性的重要决定因素。咪唑结合的Kd从pH 6时的0.7 mM增加到pH 8时的3.0 mM，并且可以拟合到pKa为7.4的单质子电离曲线，证明了咪唑鎓离子（pKa = 7.3）的优先结合。通过X射线晶体学分析验证了多种取代咪唑与W191G空腔的结合。在浸泡于1 mM 1,2-二甲基咪唑的W191G晶体中观察到最清晰的密度，这与一种定向占据一致，其中未取代的氮与天冬氨酸-235形成氢键或离子对相互作用。因此，带正电荷分子结合增强可能是与该羧酸盐相互作用的结果。类似的相互作用可能稳定野生型酶色氨酸-191自由基上正在形成的正电荷。虽然W191G的高铁中间体氧化咪唑既未预期也未观察到，但本研究确定了小分子与血红素中心附近人工创建的空腔结合的结构决定因素，该空腔能够在超过1 V的电位下产生氧化物种，这些结果将指导未来通过细胞色素c过氧化物酶进行新型底物氧化的尝试。
